Although enormous progress has been made in mapping transcription factor binding sites throughout the genome and continuing to expand our knowledge of the global binding patterns of transcription factors is very important, simply collecting genome-wide datasets will not be sufficient to answer all crucial questions about genome biology. A number of methodological problems now need to be addressed. In particular, we must develop new methods for the functional analysis of complex regulatory elements. We have identified regions of the genome that are bound by a large number of transcription factors but are not likely to be promoter regions because they are located far from all known genes, predicted genes, expressed RNAs, and H3K4me3 signals; we have called these regions chromatin hubs. We propose to develop a method by which the hubs can be investigated in their normal genomic context. Most approaches designed to study the function of a complex regulatory element involve reporter analyses and cannot reveal effects on long-range regulation. In contrast, our technology will be based on pairs of zinc finger nucleases (ZFNs) and/or transcription activator-like effector nucleases (TALENs) that will allow precise deletion of critical regulatory regions of the human genome. In essence, we will develop an approach that will allow the study of a complex element by specifically deleting it from the human genome, thus enabling analysis of an entire set of closely packed transcription factor binding sites in ther natural genomic context. Our technology will not only be invaluable for the study of chromatin hubs (as described in our proposal) but will also be appropriate for an in situ characterization of the function of any complex regulatory region. Recent genomic research has focused on documenting expressed transcripts and identifying transcription factor binding sites. The next phase of genomics requires the development of technologies that will allow the investigator to understand how cis regulatory regions influence the transcriptome. Our studies will help to integrate different types of genomic data and provide new insights into genomic regulation.